Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells—existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V–microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10 6 Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels